Neuronal overexpression of hTDP-43 in Caenorhabditis elegans impairs motor function

Transactive response DNA binding-protein 43 (TDP-43) is a conserved RNA/DNA-binding protein with a role in RNA metabolism and homeostasis. Aberrant TDP-43 functioning has been considered a major culprit in amyotrophic lateral sclerosis (ALS). Caenorhabditis elegans can be used to phenocopy ALS in vivo . Since disrupted locomotion is a strong readout of toxicity, we examined multiple motor phenotypes of a C. elegans model expressing human wild-type TDP-43 ( hTDP-43 ) pan-neuronally. Our data reveal that impaired locomotion includes more than the common deficits in crawling capacity and the presence of early-onset paralysis. We show that reduced thrashing, abnormal coiling, and decreased pharyngeal pumping are also observed, in a temperature-dependent fashion.


Description
After observing TDP-43 pathology in a Caenorhabditis elegans model expressing human TDP-43 pan-neuronally (hTDP-43 worms) (Koopman et al., 2023a) we investigated the functional consequences of these molecular features by assessing global motor function (Figure 2A). Movement phenotypes like crawling and thrashing have proven to be auxiliary in studies concerning the pathology of muscles and neurons (Brignull et al., 2006;Sorrentino et al., 2006;Ash et al., 2010;van Ham et al., 2010;Hahm et al., 2015). Due to differences in kinematics, the pattern of muscle activity and involvement of sensory neurons, trashing (i.e. the frequency of lateral bends in liquid, c-shapes) and crawling (e.g. the two-dimensional sinusoidal wave-like movement, s-shapes) provide distinct but complementary information about the (neuromuscular) health of C. elegans (Karbowski et al., 2006;Korta et al., 2007;Pierce-Shimomura et al., 2008;Hahm et al., 2015;Fang-Yen et al., 2015). In addition, rhythmic movements that are dependent on interactions between the nervous system and muscles can also be studied by assessing the pharyngeal pumping rate (Trojanowski et al., 2016). Pharyngeal pumping rates decrease with age and provide another fitness parameter for C. elegans (Chow et al., 2006).
Using automated tracking we found that the crawling capacity of hTDP-43 animals, expressed as average and maximal translocation speed, was impeded ( Figure 2B-D). The slow movement of hTDP-43 worms was accompanied by a distinctive 'uncoordinated' phenotype as previously described (Ash et al., 2010). Similarly, thrashing frequency and consequential translocation were reduced by the presence of hTDP-43 (Figure 2E-G). Here, impaired 'thrashing' coincided with an 'coiler' phenotype ( Figure 2H), a posture that is often observed in mutants with abnormal cholinergic signaling (Rand et al., 1984;Sluder et al., 2012). Given the large effects of TDP-43 on locomotion, the decrease in pharyngeal pumping appeared relatively moderate ( Figure 2I). Impaired locomotion early in life progressed to complete paralysis at adulthood day 9 (20 °C) or day 6 (25 °C) ( Figure 2J). Noteworthy, the mechanical stimuli provided to assess paralysis revealed a clear 'shrinker' phenotype characteristic for GABAergic dysfunction (Schuske et al., 2004). Together, a higher environmental temperature consistently enhanced most of the phenotypic abnormalities of hTDP-43 worms. Importantly, similar temperature-dependent effects, but to a lesser extent than those seen in hTDP-43 worms, were observed in control worms (Figure 2C-I).
In conclusion, our data shows that neuronal expression of TDP-43 impairs locomotor function in C. elegans. Moreover, the existence of more specific features such as coiling, touch-induced shrinking and uncoordinated movement provide insights into the neuronal systems affected by TDP-43.

Strains and maintenance
Standard conditions were used for C. elegans propagation at 20 °C (Brenner, 1974). Animals were age-synchronized by hypochlorite bleaching and subsequently allowed to hatch overnight in M9 buffer at 20 °C. For experiments age synchronized L1s were cultured for 72h at either 20 °C or 25 °C on NGM plates seeded with OP50 before being tested, unless stated differently.

Thrashing and crawling
For crawling ability, we recorded the free crawling of worms on empty NGM agar plates for 30 seconds at 20 fps (except for the generation of crawling maps, for which worms were recorded for 10 min. at 3 fps) with the WF-NTP platform. Determination of crawling speed and generation of crawling maps was performed using the WF-NTP software (Koopman et al., 2020). Data on thrashing were acquired by transferring worms to empty NGM agar plates flooded with M9 and subsequently recording them for 30 seconds at 20 fps. The WF-NTP software was used to derive the thrashing frequency, but also changes in eccentricity. Since uncoordinated worms do skew centroid-based trackers, we additionally recounted (at least) one experiment manually by counting at least 15 randomly chosen worms per video.

Paralysis
Synchronized worms were cultured on standard NGM plates seeded with OP50 at 20 °C. At day 1 of adulthood, 50 worms per condition (unless stated differently) were transferred to 6-cm NGM plates containing FUdR (10 worms per plate). Plates were kept at 20 °C or 25 °C, as described in the figures. Animals were tested for paralysis every day by tapping their nose/tail with a platinum wire as described previously (Cohen et al., 2006). Worms that failed to show a touch-response (i.e., moving their nose, but not their body) were scored as paralyzed. Worms that did not move, did not show a touch-response, and had no pharyngeal pumping were considered dead and were excluded from analysis.

Pharyngeal pumping
Worms were freely moving on freshly seeded NGM plates and were examined under an optical microscope to determine pumping rates by visual observations. One pharyngeal pump was defined as a complete forward and backward movement of the grinder in the pharynx (Hobson et al., 2006). The rate was counted for 30 seconds.